Professor Robert Dunn and his group at the University of Kansas are funded to develop innovative, cost-effective approaches for rapidly analyzing complex mixtures. Since the analysis of complex mixtures is central to fields such as medical diagnostics and food analysis, the development of inexpensive approaches that improve performance and reduce analysis times can have a significant impact. Dr. Dunn and his team are developing separation methods based on capillary electrophoresis, which uses strong electric fields in small capillaries to separate analytes based on their differing charge-to-size ratios. Laser-based methods for sensitive refractive index detection are being developed that can detect all analytes from small inorganic ions to large proteins without the use of labels or additional contrast agents. This simplifies the instrumentation, streamlines assay workflow, and improves the speed of analysis. In this project, the team will push detection limits down to the single molecule level by leveraging recent advancements in single molecule scattering measurements. This approach not only improves label-free detection to the ultimate limit, but recent work has shown that scattering signals are correlated with molecular mass for large macromolecules. Graduate and undergraduate students working in a highly collaborative environment, therefore, will integrate this detection method with capillary electrophoresis to create an all-optical mass detector for rapid separations.
One novel aspect of the electrophoretic separation platform developed in the Dunn lab revolves around the size of the fused silica capillary used for separations. A short 10 cm total length capillary leads to rapid separations while the closely matched inner (50 microns) and outer (80 microns) diameters results in a very thin capillary wall (15 microns). The thin wall improves heat dissipation and reduces the deleterious effects of Joule heating. It also enables the use of high numerical aperture (NA) objective lens, with very short working distances, to be integrated with the detection zone of the capillary. This is key for single molecule scattering measurements since high NA optics are required to collect the high angle scattering from a single macromolecule. The thin wall also leads to very flexible capillaries. Because of this flexibility, longer 30 cm capillaries can be looped to bring the separating species past the detector three times. This multi-pass configuration will be developed to combine separation and mass photometry measurements in the first pass followed by subsequent Taylor dispersion analysis of hydrodynamic radii in the final two passes. The fully integrated analysis platform, therefore, will be capable of quantifying charge, mass and hydrodynamic radii of separated species in one consolidated assay.
This award reflects NSF’s statutory mission and has been deemed worthy of support through evaluation using the Foundation’s intellectual merit and broader impacts review criteria.